Soft, Dynamic Hydrogel Confinement Improves Kidney Organoid Lumen Morphology and Reduces Epithelial-Mesenchymal Transition in Culture.
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ABSTRACT:
SUBMITTER: Ruiter FAA
PROVIDER: S-EPMC9284132 | biostudies-literature |
REPOSITORIES: biostudies-literature
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The lyophilized ECM powder was split into 3 biological replicates, each processed independently and analyzed in triplicate by LC-MS/MS. The powder was resuspended in lysis buffer and two spike-in proteins (each at 0.5 µg/100 µg of protein powder) were added: carnitine monooxygenase oxygenase subunit (cntA, D0C9N6) from Acinetobacter baumannii, and CTP synthase (CTPsyn, Q9VUL1) from Drosophila melanogaster. Protein extraction was performed by heating at 90°C for 10 min, and centrifuging at maximum velocity for 10 min at 4°C. ECM-derived proteins were reduced in 0.1 M dithiothreitol (DTT) at 95°C for 5 min, dissolved in 8 M urea solution after cooling down to room temperature, alkylated with 55 mM iodoacetamide for 30 min at 25°C in the dark. Alkylated proteins were purified using Microcon YM-10 filter unit for 8 times at 14000g for 40min followed by trypsin digestion for 16 h at 37°C. pH was adjusted to 3 by addition of formic acid. Peptides were desalted by C-18 column and dried into powder and were then re-suspended in 30 µl 0.1% acetic acid for the following mass spectrometry analysis.
Protein identification by liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed using Thermo Fusion Mass Spectrometer with Thermo Easy-nLC1000 Liquid Chromatography. 130 min of LC-MS gradients were performed by increasing organic proportion. The first level of MS was detected by Orbitrap with parameter of Resolution at 120K, Scan Rang at 300-1800 m/z, Mass Tolerance at 10 ppm. The second level of MS was isolated by Quadrupole, activated by HCD and detected by Orbitrap. The Orbitrap Resolution for the second level of MS was 30K.
Peak files .mzXML were obtained by RawConverter starting from .raw files.
Starting from peak files, the mass spectrometry–derived data were searched against a human protein database (Uniprot Homo sapiens reference proteome, UP000005640) by MaxQuant v. 1.6.7.0. Oxidation of methionine residues and acetyl of protein N-term were set as variable modifications. Carbamidomethyl on cysteine was set as fixed modification. Peptide-spectrum matches (PSMs) were adjusted to a 1% and then assembled further to a final protein-level false discovery rate (FDR) of 1%.
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